Ura5 gene and methods for stable genetic integration in yeast

ABSTRACT

A novel gene encoding  P. pastoris  orotate-phosphoribosyl transferase (URA5) is disclosed. Methods for producing and selecting yeast strains capable of stable genetic integration of heterologous sequences into the host genome are also provided.

FIELD OF THE INVENTION

This invention relates to novel genes isolated in yeast. The inventionalso relates to plasmids, which are particularly useful for stablegenetic integration into the yeast genome. The present invention alsorelates to novel yeast strains in the expression of heterologousproteins and to methods of generating the novel strains.

BACKGROUND OF THE INVENTION

Yeast strains, such as Pichia pastoris, are commonly used for theproduction of heterologous proteins. P. pastoris has become a popularmodel system for the study of peroxisome biogenesis (Gould et al., Yeast8:613-628 (1992)), autophagy (Tuttle and Dunn, J. Cell Sci. 108:25-35(1995); Sakai et al., J. Cell Biol. 141:625-636 (1998)) and theorganization and biogenesis of the organelles of the secretory pathway(Rossanese et al., J. Cell Biol. 145:69-81 (1999)). The development ofsimple DNA transformation systems (see Cregg et al., Mol. Cell. Biol.5:3376-3385 (1985)) and the availability of selectable marker genes havebeen of great importance in conducting the above experiments. Currently,the biosynthetic marker genes ADE1, ARG4, HIS4 and URA3 are used inconjunction with the corresponding auxotrophic host strains to selectfor transformed cells. Lin Cereghino et al., Gene 263:159-169 (2001).The use of dominant selectable markers to identify transformants is alsopossible, but markers are limited to the Sh ble gene fromStreptoalloteichus hindustanus, which confers resistance to the drugZeocin (Higgins et al., Methods Mol. Biol. 103:41-53 (1998)), and theblasticidin S deaminase gene from Aspergillus terreus, which confersresistance to the drug blasticidin (Kimura et al., Mol. Gen. Genet.242:121-129 (1994)).

Stable integration of cloned DNA segments into the yeast genome throughhomologous recombination is well known in the art. See e.g., Orr-Weaveret al., Proc. Natl. Acad. Sci. USA 78:6364-6358 (1981). More recently,methods have been developed in S. cerevisiae to generate yeast strainscontaining DNA integrated at multiple unlinked sites by homologousrecombination using molecular constructs containing the URA3 markergene. See e.g., Alani et al., Genetics 116: 541-545 (1987). In thisapproach, a construct is generated in which the S. cerevisiae URA3 geneis flanked by direct repeats of a Salmonella hisG DNA. This construct isinserted into a cloned target gene of interest and the linear cassette,containing the complete URA3 gene flanked by direct repeats from hisGand further flanked by 5′ and 3′ segments from the target gene, isintroduced into a Ura3⁻ mutant yeast strain by transformation. Stableintegrants arising from homologous recombination at the genomic locus ofthe target gene linked to the URA3 marker gene are then isolated byselection for growth in the absence of uracil. Excision of the URA3 genethrough a recombination event between the flanking hisG direct repeatsegments restores uracil auxotrophy (Ura⁻) but leaves behind a disruptedgenomic copy of the target gene. Ura⁺ strains of S. cerevisiae areunable to grow on medium supplemented with the pyrimidine analog5-fluoroorotic acid (5-FOA) whereas Ura⁻ cells survive such treatment.Cells lacking the URA3 gene are thus readily identified using a positivecounterselection on 5-FOA. Boeke et al., Mol. Gen. Genet. 197:345-346(1984). Through repeated use of the recyclable URA3 marker construct,multiple different genes of interest can be disrupted within a singlestrain. Similar approaches have been used in other fungi. Wilson et al.,Yeast 16:65-70 (2000).

Extensive genetic engineering projects, where several genes in parallelhave to be expressed and several others have to be eliminated, requirethe use of counterselectable markers and plasmids for stable geneticintegration of heterologous proteins into the host genome. Recently, anew counterselectable marker based on the T-urf13 gene from themitochondrial genome of male-sterile maize has been described for P.pastoris. Soderholm et al., BioTechniques 31:306-312 (2001). Toxicity ofthe T-wf13 gene appears to be a host specific problem, however, as thegene may be conditionally lethal with certain gene disruptions that areotherwise not lethal. In addition, the gene is also toxic in the absenceof the counterselecting agent methomyl, therefore, the counterselectionstep must be performed immediately. In addition, a separate gene isrequired for the initial positive selection step, and the agent used forthe counterselection step, methomyl, is light sensitive and breaks downrapidly in aqueous solutions. The system is therefore more complicatedthan the URA3 system described above, in which the same gene isresponsible both for the initial selection of Ura⁺ prototrophs and forthe subsequent counterselection of Ura⁻ auxotrophs. It would be usefulto find new marker genes in the yeast pyrimidine biosynthetic pathway inwhich selection of auxotrophs and counterselection using 5-FOA orsimilarly acting agents may be used to select and counterselect a singlemarker gene in multiple rounds of genetic transformation at differentloci.

Five structural genes providing six enzymatic steps are responsible forendogenous pyrimidine biosynthesis in S. cerevisiae. Montigny et al.,Mol. Gen. Genet. 215:455-462 (1989). The last two steps in this pathway,the conversion of orotic acid to orotidine 5′-phosphate and theconversion of orotidine 5′-phosphate to uridine 5′-phosphate, arecatalyzed by orotate phosphoribosyltransferase (OPRTase) andorotidine-5′-phosphate decarboxylase (OMPdecase). These enzymes areencoded by the URA5 gene and the URA3 gene, respectively. Both geneshave been cloned, characterized and used for genetic integration in S.cerevisiae, but only the URA3 gene has been cloned in P. pastoris.

The S. cerevisiae URA5 gene was cloned by complementation of anon-reverting E. coli pyrE mutant that was blocked inorotate-phosphoribosyl transferase activity. Montigny et al., Mol. Gen.Genet. 215:455-462 (1989). Yeast cells lacking this gene displayed aleaky phenotype, however, indicating that, in S. cerevisiae, anotherprotein possesses orotate-phosphoribosyl transferase activity. See Jundand Lacroute, J. Bacteria 109:196-202 (1972). The URA5 gene has alsobeen identified in Kluyveromyces lactis. Bai et al., Yeast 15:1393-1398(1999). The gene order around the URA5 gene has been examined in S.cerevisiae, K. lactis, C. albicans and Y. lipolytica. Sánchez andDomínguez, Yeast 18:807-813 (2001). In all four organisms, the URA5 geneand a gene which functions in the secretory pathway (the SEC65 gene) arearranged adjacent to one another and in the opposite relativeorientation.

A selection system based on disrupting the URA3 gene in P. pastoris hasrecently been disclosed. U.S. Pat. No. 6,051,419. The methods describedtherein also provide “pop-in” (site-directed integration of thetransforming DNA by gene addition) and “pop-out” (recombination betweenfunctional and nonfunctional genes resulting in the loss of one of thesegenes and the URA3 gene) in what is referred to as a “bidirectionalselection process.” “Pop-in/pop-out” gene replacement using S.cerevisiae URA3 is a convenient method because, as described above, theselection marker can be recycled. See Boeke et al., Meth. Enzymol.154:164-175 (1987). P. pastoris ura3 auxotrophs, however, grow slowly.U.S. Pat. No. 6,051,419. In addition, because the sequences responsiblefor homologous recombination in the “popping out” step are the same asthose responsible for the “popping in” step in a single-crossoverrecombination process, the genetic material inserted by “pop-in” islikely to be lost by “pop-out”. The method is thus more suitable forgenerating point mutants or gene disruptions than for stablyincorporating expressable heterologous genes of interest into thegenome.

Currently available auxotrophic strains of P. pastoris suffer thefurther disadvantage that the respective auxotrophic marker genes havethe potential to revert. A high reversion rate decreases the usefulnessof auxotrophic strains, because revertant colonies are misidentified asfalse-positive transformants.

Given the utility of the URA3 selection and counterselection system inS. cerevisiae and the limitations on using these and other currentmethods in other yeast and fungi, the identification of a URA5 gene inP. pastoris and the development of a system for selecting stable geneticintegration events using URA5 would be useful.

SUMMARY OF THE INVENTION

The present invention provides isolated polynucleotides comprising orconsisting of nucleic acid sequences selected from the group consistingof the coding sequences of the P. pastoris URA5 gene, a fragment of theP. pastoris SEC65 gene and a fragment of the P. pastoris SCS7 gene;nucleic acid sequences that are degenerate variants of these sequences;and related nucleic acid sequences and fragments. The invention alsoprovides vectors and host cells comprising these isolatedpolynucleotides.

The invention further provides isolated polypeptides comprising orconsisting of polypeptide sequences selected from the group consistingof sequences encoded by the P. pastoris URA5 gene, by a fragment of theP. pastoris SEC65 gene and by a fragment of the P. pastoris SCS7 gene,and related polypeptide sequences, fragments and fusions. Antibodiesthat specifically bind to the isolated polypeptides of the invention arealso provided.

The invention also provides host cells comprising a disruption, deletionor mutation of a nucleic acid sequence selected from the groupconsisting of the coding sequence of the P. pastoris URA5 gene, anucleic acid sequence that is a degenerate variant of the codingsequence of the P. pastoris URA5 gene and related nucleic acid sequencesand fragments, in which the host cells have a reduced activity of thepolypeptide encoded by the nucleic acid sequence compared to a host cellwithout the disruption, deletion or mutation.

The invention further provides methods for the genetic integration of aheterologous nucleic acid sequence in a host cell. These methodscomprise the step of disrupting a host gene encodingorotate-phosphoribosyl transferase by introduction of a disrupted,deleted or mutated nucleic acid sequence derived from a sequenceselected from the group consisting of the coding sequence of the P.pastoris URA5 gene, a nucleic acid sequence that is a degenerate variantof the coding sequence of the P. pastoris URA5 gene and related nucleicacid sequences and fragments.

In addition, the invention provides methods for the genetic integrationof a heterologous nucleic acid sequence in a host cell lackingorotate-phosphoribosyl transferase activity. These methods comprise thestep of introducing a sequence of interest into the host cell in linkagewith a sequence encoding orotate-phosphoribosyl transferase activityselected from the group consisting of the coding sequence of the P.pastoris URA5 gene, a nucleic acid sequence that is a degenerate variantof the coding sequence of the P. pastoris URA5 gene and related nucleicacid sequences and fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a 1947 bp URA5-containing genomic fragment (Sau3A-SspI) ofP. pastoris (SEQ ID NO:1), including the URA5 coding sequence (SEQ IDNO:2) and its encoded polypeptide (SEQ ID NO:3), the sequencecomplementary to the 3′ fragment of the SEC65 coding sequence (SEQ IDNO:4) and its encoded polypeptide (SEQ ID NO:5), and the 3′ fragment ofthe SCS7 coding sequence (SEQ ID NO:6) and its encoded polypeptide (SEQID NO:7).

FIG. 2 shows an alignment of sequences used to design degenerateprimers. The URA5-related sequences are URA5 from S. cerevisiae (SEQ IDNO:8), URA10 from S. cerevisiae (SEQ ID NO:9), and URA5 from K. lactis(SEQ ID NO:10), Y. lipolytica (SEQ ID NO:11), S. pombe (SEQ ID NO:12),T. reesei (SEQ ID NO:13), E. coli (SEQ ID NO:14), P. aeruginosa (SEQ IDNO:15), and H. influenzae (SEQ ID NO:16). The URA5 sequence from P.pastoris (residues 27-80 of SEQ ID NO:3) is shown for comparison. TheSEC65-related sequences are from S. cerevisiae (SEQ ID NO:17), K. lactis(SEQ ID NO:18), C. albicans (SEQ ID NO:19), Y. lipolytica (SEQ IDNO:20), N. crassa (SEQ ID NO:21), and S. pombe (SEQ ID NO:22).

FIG. 3 depicts some of the degenerate oligonucleotides used in cloningof the P. pastoris URA5 gene. These oligonucleotides are URA5-1 (SEQ IDNO:23), URA5-2 (SEQ ID NO:24), URA5-3 (SEQ ID NO:25), URA5-4 (SEQ IDNO:26), URA5-5 (SEQ ID NO:27), and URA5-6 (SEQ ID NO:28).

FIG. 4 shows restriction maps of plasmid pJN266 (including a recyclableURA3 cassette, which may be used to disrupt a KEX1 locus); plasmidpJN315 (including the P. pastoris URA3 gene flanked by lacZ directrepeats); and plasmid pJN329 (including a recyclable URA3 cassette,which may be used to disrupt an OCH1 locus).

FIG. 5 shows restriction maps of plasmid pJN395 (including a P. pastorisURA5 disruption cassette marked with a kanamycin-resistance gene);plasmid pJN396 (including the P. pastoris URA5 gene flanked by lacZdirect repeats); plasmid pJN398 (including a recyclable URA5 cassette,which may be used to knock out an OCH1 locus); and plasmid pJN407(including a P. pastoris URA5-K. lactis UDP-GlcNAc Transporter cassette,which may be used for stable integration into an OCH1 locus).

FIG. 6 shows the use of a P. pastoris URA5-K. lactis UDP-GlcNAcTransporter cassette in the stable integration of the UDP-GlcNAcTransporter into the OCH1 locus.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall include theplural and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of biochemistry,enzymology, molecular and cellular biology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art. The methods andtechniques of the present invention are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated. See,e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989);Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992, and Supplements to 2002); Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction toGlycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual,Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry:Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry:Section A Proteins, Vol II, CRC Press (1976); Essentials ofGlycobiology, Cold Spring Harbor Laboratory Press (1999).

All publications, patents and other references mentioned herein arehereby incorporated by reference in their entireties.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-nativeinternucleoside bonds, or both. The nucleic acid can be in anytopological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked conformation.

Unless otherwise indicated, a “nucleic acid comprising SEQ ID NO:X”refers to a nucleic acid, at least a portion of which has either (i) thesequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQ IDNO:X. The choice between the two is dictated by the context. Forinstance, if the nucleic acid is used as a probe, the choice between thetwo is dictated by the requirement that the probe be complementary tothe desired target.

An “isolated” or “substantially pure” nucleic acid or polynucleotide(e.g., an RNA, DNA or a mixed polymer) is one which is substantiallyseparated from other cellular components that naturally accompany thenative polynucleotide in its natural host cell, e.g., ribosomes,polymerases and genomic sequences with which it is naturally associated.The term embraces a nucleic acid or polynucleotide that (1) has beenremoved from its naturally occurring environment, (2) is not associatedwith all or a portion of a polynucleotide in which the “isolatedpolynucleotide” is found in nature, (3) is operatively linked to apolynucleotide which it is not linked to in nature, or (4) does notoccur in nature. The term “isolated” or “substantially pure” also can beused in reference to recombinant or cloned DNA isolates, chemicallysynthesized polynucleotide analogs, or polynucleotide analogs that arebiologically synthesized by heterologous systems.

However, “isolated” does not necessarily require that the nucleic acidor polynucleotide so described has itself been physically removed fromits native environment. For instance, an endogenous nucleic acidsequence in the genome of an organism is deemed “isolated” herein if aheterologous sequence is placed adjacent to the endogenous nucleic acidsequence, such that the expression of this endogenous nucleic acidsequence is altered. In this context, a heterologous sequence is asequence that is not naturally adjacent to the endogenous nucleic acidsequence, whether or not the heterologous sequence is itself endogenous(originating from the same host cell or progeny thereof) or exogenous(originating from a different host cell or progeny thereof). By way ofexample, a promoter sequence can be substituted (e.g., by homologousrecombination) for the native promoter of a gene in the genome of a hostcell, such that this gene has an altered expression pattern. This genewould now become “isolated” because it is separated from at least someof the sequences that naturally flank it.

A nucleic acid is also considered “isolated” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “isolated” if it contains an insertion, deletion or a pointmutation introduced artificially, e.g., by human intervention. An“isolated nucleic acid” also includes a nucleic acid integrated into ahost cell chromosome at a heterologous site and a nucleic acid constructpresent as an episome. Moreover, an “isolated nucleic acid” can besubstantially free of other cellular material, or substantially free ofculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence. The term “degenerate oligonucleotide” or “degenerate primer”is used to signify an oligonucleotide capable of hybridizing with targetnucleic acid sequences that are not necessarily identical in sequencebut that are homologous to one another within one or more particularsegments.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFASTA, Gap or Bestfit, which are programs in Wisconsin Package Version10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. Pearson, MethodsEnzymol. 183:63-98 (1990) (hereby incorporated by reference in itsentirety). For instance, percent sequence identity between nucleic acidsequences can be determined using FASTA with its default parameters (aword size of 6 and the NOPAM factor for the scoring matrix) or using Gapwith its default parameters as provided in GCG Version 6.1, hereinincorporated by reference. Alternatively, sequences can be comparedusing the computer program, BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 50%, more preferably 60%of the nucleotide bases, usually at least about 70%, more usually atleast about 80%, preferably at least about 90%, and more preferably atleast about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap, as discussed above.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof hybridizes to another nucleic acid, to a strandof another nucleic acid, or to the complementary strand thereof, understringent hybridization conditions. “Stringent hybridization conditions”and “stringent wash conditions” in the context of nucleic acidhybridization experiments depend upon a number of different physicalparameters. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, solvents, the basecomposition of the hybridizing species, length of the complementaryregions, and the number of nucleotide base mismatches between thehybridizing nucleic acids, as will be readily appreciated by thoseskilled in the art. One having ordinary skill in the art knows how tovary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (T_(m)) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference.For purposes herein, “stringent conditions” are defined for solutionphase hybridization as aqueous hybridization (i.e., free of formamide)in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1%SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1%SDS at 65° C. for 20 minutes. It will be appreciated by the skilledworker that hybridization at 65° C. will occur at different ratesdepending on a number of factors including the length and percentidentity of the sequences which are hybridizing.

The nucleic acids (also referred to as polynucleotides) of thisinvention may include both sense and antisense strands of RNA, cDNA,genomic DNA, and synthetic forms and mixed polymers of the above. Theymay be modified chemically or biochemically or may contain non-naturalor derivatized nucleotide bases, as will be readily appreciated by thoseof skill in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such asuncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.) Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule. Other modifications can include, for example, analogs in whichthe ribose ring contains a bridging moiety or other structure such asthe modifications found in “locked” nucleic acids.

The term “mutated” when applied to nucleic acid sequences means thatnucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989)and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and“oligonucleotide-directed mutagenesis” (a process which enables thegeneration of site-specific mutations in any cloned DNA segment ofinterest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57(1988)).

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments may beligated into the viral genome (discussed in more detail below). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., vectors having an origin of replication whichfunctions in the host cell). Other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and arethereby replicated along with the host genome. Moreover, certainpreferred vectors are capable of directing the expression of genes towhich they are operatively linked. Such vectors are referred to hereinas “recombinant expression vectors” (or simply, “expression vectors”).

As used herein, the term “sequence of interest” or “gene of interest”refers to a nucleic acid sequence, typically encoding a protein, that isnot normally produced in the host cell. The methods disclosed hereinallow one or more sequences of interest or genes of interest to bestably integrated into a host cell genome. Non-limiting examples ofsequences of interest include sequences encoding one or morepolypeptides having an enzymatic activity, e.g., an enzyme which affectsN-glycan synthesis in a host such as mannosyltransferases,N-acetylglucosaminyltransferases, UDP-N-acetylglucosamine transporters,galactosyltransferases and sialyltransferases. Other non-limitingexamples include sequences encoding one or more polypeptides having anenzymatic activity, e.g., an enzyme which affects O-glycan synthesis ina host such as protein-mannosyltransferase (PMT) genes. Still othersequences encode proteins of interest such as kringle domains of thehuman plasminogen, erythropoietin, cytokines such as interferon-α,interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF,coagulation factors such as factor VIII, factor IX, and human protein C,soluble IgE receptor α-chain, IgG, IgG fragments, IgM, urokinase,chymase, and urea trypsin inhibitor, IGF-binding protein, epidermalgrowth factor, growth hormone-releasing factor, annexin V fusionprotein, angiostatin, vascular endothelial growth factor-2, myeloidprogenitor inhibitory factor-1, osteoprotegerin, α-1 antitrypsin, DNaseII and α-feto proteins.

The term “marker sequence” or “marker gene” refers to a nucleic acidsequence capable of expressing an activity that allows either positiveor negative selection for the presence or absence of the sequence withina host cell. For example, the P. pastoris URA5 gene is a marker genebecause its presence can be selected for by the ability of cellscontaining the gene to grow in the absence of uracil. Its presence canalso be selected against by the inability of cells containing the geneto grow in the presence of 5-FOA. Marker sequences or genes do notnecessarily need to display both positive and negative selectability.Non-limiting examples of marker sequences or genes from P. pastorisinclude ADE1, ARG4, HIS4 and URA3.

“Operatively linked” expression control sequences refers to a linkage inwhich the expression control sequence is contiguous with the gene ofinterest to control the gene of interest, as well as expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” encompasses both naturally-occurring andnon-naturally-occurring proteins, and fragments, mutants, derivativesand analogs thereof. A polypeptide may be monomeric or polymeric.Further, a polypeptide may comprise a number of different domains eachof which has one or more distinct activities.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) exists in a purity not found in nature, wherepurity can be adjudged with respect to the presence of other cellularmaterial (e.g., is free of other proteins from the same species) (3) isexpressed by a cell from a different species, or (4) does not occur innature (e.g., it is a fragment of a polypeptide found in nature or itincludes amino acid analogs or derivatives not found in nature orlinkages other than standard peptide bonds). Thus, a polypeptide that ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be “isolated” fromits naturally associated components. A polypeptide or protein may alsobe rendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.As thus defined, “isolated” does not necessarily require that theprotein, polypeptide, peptide or oligopeptide so described has beenphysically removed from its native environment.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has a deletion, e.g., an amino-terminal and/or carboxy-terminaldeletion compared to a full-length polypeptide. In a preferredembodiment, the polypeptide fragment is a contiguous sequence in whichthe amino acid sequence of the fragment is identical to thecorresponding positions in the naturally-occurring sequence. Fragmentstypically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferablyat least 12, 14, 16 or 18 amino acids long, more preferably at least 20amino acids long, more preferably at least 25, 30, 35, 40 or 45, aminoacids, even more preferably at least 50 or 60 amino acids long, and evenmore preferably at least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof thatare substantially homologous in primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those skilledin the art.

A variety of methods for labeling polypeptides and of substituents orlabels useful for such purposes are well known in the art, and includeradioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H, ligands which bindto labeled antiligands (e.g., antibodies), fluorophores,chemiluminescent agents, enzymes, and antiligands which can serve asspecific binding pair members for a labeled ligand. The choice of labeldepends on the sensitivity required, ease of conjugation with theprimer, stability requirements, and available instrumentation. Methodsfor labeling polypeptides are well known in the art. See, e.g., Ausubelet al., Current Protocols in Molecular Biology, Greene PublishingAssociates (1992, and Supplements to 2002) (hereby incorporated byreference).

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 100 or 125 amino acids. Fusions that include theentirety of the proteins of the present invention have particularutility. The heterologous polypeptide included within the fusion proteinof the present invention is at least 6 amino acids in length, often atleast 8 amino acids in length, and usefully at least 15, 20, and 25amino acids in length. Fusions that include larger polypeptides, such asan IgG Fc region, and even entire proteins, such as the greenfluorescent protein (“GFP”) chromophore-containing proteins, haveparticular utility. Fusion proteins can be produced recombinantly byconstructing a nucleic acid sequence which encodes the polypeptide or afragment thereof in frame with a nucleic acid sequence encoding adifferent protein or peptide and then expressing the fusion protein.Alternatively, a fusion protein can be produced chemically bycrosslinking the polypeptide or a fragment thereof to another protein.

As used herein, the term “antibody” refers to a polypeptide, at least aportion of which is encoded by at least one immunoglobulin gene, orfragment thereof, and that can bind specifically to a desired targetmolecule. The term includes naturally-occurring forms, as well asfragments and derivatives.

Fragments within the scope of the term “antibody” include those producedby digestion with various proteases, those produced by chemical cleavageand/or chemical dissociation and those produced recombinantly, so longas the fragment remains capable of specific binding to a targetmolecule. Among such fragments are Fab, Fab′, Fv, F(ab′)₂, and singlechain Fv (scFv) fragments.

Derivatives within the scope of the term include antibodies (orfragments thereof) that have been modified in sequence, but remaincapable of specific binding to a target molecule, including:interspecies chimeric and humanized antibodies; antibody fusions;heteromeric antibody complexes and antibody fusions, such as diabodies(bispecific antibodies), single-chain diabodies, and intrabodies (see,e.g., Intracellular Antibodies: Research and Disease Applications,(Marasco, ed., Springer-Verlag New York, Inc., 1998), the disclosure ofwhich is incorporated herein by reference in its entirety).

As used herein, antibodies can be produced by any known technique,including harvest from cell culture of native B lymphocytes, harvestfrom culture of hybridomas, recombinant expression systems and phagedisplay.

The term “non-peptide analog” refers to a compound with properties thatare analogous to those of a reference polypeptide. A non-peptidecompound may also be termed a “peptide mimetic” or a “peptidomimetic”.See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford UniversityPress (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: AHandbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry—APractical Textbook, Springer Verlag (1993); Synthetic Peptides: A UsersGuide, (Grant, ed., W. H. Freeman and Co., 1992); Evans et al., J. Med.Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veberand Freidinger, Trends Neurosci., 8:392-396 (1985); and references sitedin each of the above, which are incorporated herein by reference. Suchcompounds are often developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar to usefulpeptides of the invention may be used to produce an equivalent effectand are therefore envisioned to be part of the invention.

A “polypeptide mutant” or “mutein” refers to a polypeptide whosesequence contains an insertion, duplication, deletion, rearrangement orsubstitution of one or more amino acids compared to the amino acidsequence of a native or wild-type protein. A mutein may have one or moreamino acid point substitutions, in which a single amino acid at aposition has been changed to another amino acid, one or more insertionsand/or deletions, in which one or more amino acids are inserted ordeleted, respectively, in the sequence of the naturally-occurringprotein, and/or truncations of the amino acid sequence at either or boththe amino or carboxy termini. A mutein may have the same but preferablyhas a different biological activity compared to the naturally-occurringprotein.

A mutein has at least 65% overall sequence homology to its wild-typecounterpart. Even more preferred are muteins having at least 70%, 75%,80%, 85% or 90% overall sequence homology to the wild-type protein. Inan even more preferred embodiment, a mutein exhibits at least 95%sequence identity, even more preferably 98%, even more preferably 99%and even more preferably 99.9% overall sequence identity. Sequencehomology may be measured by any common sequence analysis algorithm, suchas Gap or Bestfit.

Amino acid substitutions can include those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2^(nd) ed.1991), which is incorporated herein by reference. Stereoisomers (e.g.,D-amino acids) of the twenty conventional amino acids, unnatural aminoacids such as α-, α-disubstituted amino acids, N-alkyl amino acids, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand end corresponds to the amino terminal end and the right-handend corresponds to the carboxy-terminal end, in accordance with standardusage and convention.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences.) In a preferred embodiment, a homologousprotein is one that exhibits at least 65% sequence homology to the wildtype protein, more preferred is at least 70% sequence homology. Evenmore preferred are homologous proteins that exhibit at least 75%, 80%,85% or 90% sequence homology to the wild type protein. In a yet morepreferred embodiment, a homologous protein exhibits at least 95%, 98%,99% or 99.9% sequence identity. As used herein, homology between tworegions of amino acid sequence (especially with respect to predictedstructural similarities) is interpreted as implying similarity infunction.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art. See,e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (hereinincorporated by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using a measure of homology assignedto various substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild-type protein and amutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a particular polypeptide sequenceto a database containing a large number of sequences from differentorganisms is the computer program BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

Preferred parameters for BLASTp are:

Expectation value: 10 (default); Filter: seg (default); Cost to open agap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments:100 (default); Word size: 11 (default); No. of descriptions: 100(default); Penalty Matrix: BLOWSUM62.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences. Pearson,Methods Enzymol. 183:63-98 (1990) (herein incorporated by reference).For example, percent sequence identity between amino acid sequences canbe determined using FASTA with its default parameters (a word size of 2and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereinincorporated by reference.

“Specific binding” refers to the ability of two molecules to bind toeach other in preference to binding to other molecules in theenvironment. Typically, “specific binding” discriminates overadventitious binding in a reaction by at least two-fold, more typicallyby at least 10-fold, often at least 100-fold. Typically, the affinity oravidity of a specific binding reaction, as quantified by a dissociationconstant, is about 10⁻⁷ M or stronger (e.g., about 10⁻⁸ M, 10⁻⁹M or evenstronger).

The term “region” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein.

The term “domain” as used herein refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.Examples of protein domains include, but are not limited to, an Igdomain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Nucleic Acid Sequences

The present invention provides isolated nucleic acid molecules thatinclude the URA5 gene from P. pastoris and variants thereof. Thefull-length nucleic acid sequence for this gene, which encodes theenzyme orotate-phosphoribosyl transferase (OPRTase, EC 2.4.2.10), hasbeen identified and sequenced as set forth in FIG. 1. Included withinthe cloned genomic sequence (SEQ ID NO:1) is a coding sequence fororotate-phosphoribosyl transferase (SEQ ID NO:2). The encoded amino acidsequence is also set forth in FIG. 1 (SEQ ID NO:3). The URA5 gene isparticularly useful as a reuseable, selectable and counterselectablemarker.

Provided herein are nucleic acid molecules capable of promoting thestable genetic integration of heterologous genes (i.e. genes ofinterest) into a host genome. The combination of the URA5 marker andnucleic acids capable of promoting stable genetic integration enablesextensive strain modification. It will be readily apparent to a skilledartisan that the repeated use of the methods disclosed herein allowsmultiple genes to be disrupted in various loci and further allows theinsertion at these sites of any gene or genes of interest. Genesinserted by the disclosed approaches become stably integrated at aselected region in the genomic DNA of the host cells.

In one embodiment, the invention provides an isolated nucleic acidmolecule having a nucleic acid sequence comprising or consisting of awild-type P. pastoris URA5 coding sequence (SEQ ID NO:2), and homologs,variants and derivatives thereof. The invention also provides a nucleicacid molecule comprising or consisting of a sequence which is adegenerate variant of the wild-type P. pastoris URA5 gene. In a furtherembodiment, the invention provides a nucleic acid molecule comprising orconsisting of a sequence which is a variant of the P. pastoris URA5 genehaving at least 65% identity to the wild-type gene. The nucleic acidsequence can preferably have at least 70%, 75% or 80% identity to thewild-type gene. Even more preferably, the nucleic acid sequence can have85%, 90%, 95%, 98%, 99%, 99.9% or even higher identity to the wild-typegene.

In another embodiment, the nucleic acid molecule of the inventionencodes a polypeptide having the amino acid sequence of SEQ ID NO:3.Also provided is a nucleic acid molecule encoding a polypeptide sequencethat is at least 65% identical to SEQ ID NO:3. Typically the nucleicacid molecule of the invention encodes a polypeptide sequence of atleast 70%, 75% or 80% identity to SEQ ID NO:3. Preferably, the encodedpolypeptide is 85%, 90% or 95% identical to SEQ ID NO:3, and theidentity can even more preferably be 98%, 99%, 99.9% or even higher.

In another aspect, the invention provides a fragment of the SEC65 genefrom P. pastoris. This fragment, which is located downstream from and inthe opposite orientation to the URA5 gene, has been identified as setforth in FIG. 1 (SEQ ID NO:4). The amino acid sequence encoded by theSEC65 fragment is also set forth in FIG. 1 (SEQ ID NO:5). Accordingly,the present invention provides isolated nucleic acid molecules thatinclude a wild-type SEC65 gene fragment from P. pastoris and homologs,variants and derivatives thereof.

In one embodiment, the invention provides an isolated nucleic acidmolecule having a nucleic acid sequence comprising or consisting of afragment of the wild-type P. pastoris SEC65 gene (SEQ ID NO:4), andhomologs, variants and derivatives thereof. In an alternative embodimentof the invention, the nucleic acid sequence is a degenerate variant ofthe P. pastoris SEC65 gene fragment.

In a further embodiment of the invention, the nucleic acid sequence is avariant of the P. pastoris SEC65 gene fragment having at least 65%identity to the wild-type gene fragment. The nucleic acid sequence canpreferably have at least 70%, 75% or 80% identity to the wild-type genefragment. Even more preferably, the nucleic acid sequence can have 85%,90%, 95%, 98%, 99%, 99.9% or even higher identity to the wild-type genefragment.

In another embodiment, the nucleic acid molecule of the inventionencodes a polypeptide having the amino acid sequence of SEQ ID NO:5.Also provided is a nucleic acid molecule encoding a polypeptide sequencethat is at least 65% identical to SEQ ID NO:5. Typically, the nucleicacid molecule of the invention encodes a polypeptide sequence of atleast 70%, 75% or 80% identity to SEQ ID NO:5. Preferably, the encodedpolypeptide is 85%, 90% or 95% identical to SEQ ID NO:5, and theidentity can even more preferably be 98%, 99%, 99.9% or even higher.

In yet another aspect, the invention provides a fragment of the SCS7gene from P. pastoris. This fragment, which is located upstream from andin the same orientation as the URA5 gene, is identified as set forth inFIG. 1 (SEQ ID NO:6). The amino acid sequence encoded by the SCS7fragment is also set forth in FIG. 1 (SEQ ID NO:7). The presentinvention thus provides isolated nucleic acid molecules that include aP. pastoris wild-type SCS7 gene fragment and variants thereof.

In one embodiment, the invention provides an isolated nucleic acidmolecule having a nucleic acid sequence comprising or consisting of afragment of the wild-type P. pastoris SCS7 gene (SEQ ID NO:6), andhomologs, variants and derivatives thereof. In an alternative embodimentof the invention, the nucleic acid sequence is a degenerate variant ofthe P. pastoris SCS7 gene fragment.

In a further embodiment of the invention, the nucleic acid sequence is avariant of the P. pastoris SCS7 gene fragment having at least 65%identity to the wild-type gene fragment. The nucleic acid sequence canpreferably have at least 70%, 75% or 80% identity to the wild-type genefragment. Even more preferably, the nucleic acid sequence can have 85%,90%, 95%, 98%, 99%, 99.9% or even higher identity to the wild-type genefragment.

In another embodiment, the nucleic acid molecule of the inventionencodes a polypeptide having the amino acid sequence of SEQ ID NO:7.Also provided is a nucleic acid molecule encoding a polypeptide sequencethat is at least 65% identical to SEQ ID NO:7. Typically, the nucleicacid molecule of the invention encodes a polypeptide sequence of atleast 70%, 75% or 80% identity to SEQ ID NO:7. Preferably, the encodedpolypeptide is 85%, 90% or 95% identical to SEQ ID NO:7, and theidentity can even more preferably be 98%, 99%, 99.9% or even higher.

The invention also provides nucleic acid molecules that hybridize understringent conditions to the above-described nucleic acid molecules. Asdefined above, and as is well known in the art, stringent hybridizationsare performed at about 25° C. below the thermal melting point (T_(m))for the specific DNA hybrid under a particular set of conditions, wherethe T_(m) is the temperature at which 50% of the target sequencehybridizes to a perfectly matched probe. Stringent washing is performedat temperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions.

Nucleic acid molecules comprising a fragment of any one of theabove-described nucleic acid sequences are also provided. Thesefragments preferably contain at least 20 contiguous nucleotides. Morepreferably the fragments of the nucleic acid sequences contain at least25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguousnucleotides.

The nucleic acid sequence fragments of the present invention displayutility in a variety of systems and methods. For example, the fragmentsmay be used as probes in various hybridization techniques. Depending onthe method, the target nucleic acid sequences may be either DNA or RNA.The target nucleic acid sequences may be fractionated (e.g., by gelelectrophoresis) prior to the hybridization, or the hybridization may beperformed on samples in situ. One of skill in the art will appreciatethat nucleic acid probes of known sequence find utility in determiningchromosomal structure (e.g., by Southern blotting) and in measuring geneexpression (e.g., by Northern blotting). In such experiments, thesequence fragments are preferably detectably labeled, so that theirspecific hydridization to target sequences can be detected andoptionally quantified. One of skill in the art will appreciate that thenucleic acid fragments of the present invention may be used in a widevariety of blotting techniques not specifically described herein.

It should also be appreciated that the nucleic acid sequence fragmentsdisclosed herein also find utility as probes when immobilized onmicroarrays. Methods for creating microarrays by deposition and fixationof nucleic acids onto support substrates are well known in the art.Reviewed in DNA Microarrays: A Practical Approach (Practical ApproachSeries), Schena (ed.), Oxford University Press (1999) (ISBN:0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray BiochipTools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of which are incorporated herein by reference in theirentireties. Analysis of, for example, gene expression using microarrayscomprising nucleic acid sequence fragments, such as the nucleic acidsequence fragments disclosed herein, is a well-established utility forsequence fragments in the field of cell and molecular biology. Otheruses for sequence fragments immobilized on microarrays are described inGerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger,Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A PracticalApproach (Practical Approach Series), Schena (ed.), Oxford UniversityPress (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999);Microarray Biochip Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of each of which is incorporated herein by reference in itsentirety.

In another embodiment, isolated nucleic acid molecules encoding apolypeptide having orotate-phosphoribosyl transferase activity areprovided. As is well known in the art, enzyme activities can be measuredin various ways. For example, the pyrophosphorolysis of OMP may befollowed spectroscopically. Grubmeyer et al., J. Biol. Chem.268:20299-20304 (1993). Additional examples of substrates useful for thespectroscopic assay of orotate-phosphoribosyl transferase activity arealso known in the art. Shostak et al., Anal Biochem. 191:365-369 (1990).Alternatively, the activity of the enzyme can be followed usingchromatographic techniques, such as by high performance liquidchromatography. Chung and Sloan, J. Chromatogr. 371:71-81 (1986). Othermethods and techniques may also be suitable for the measurement ofenzyme activity, as would be known by one of skill in the art.

The invention also provides recombinant DNA molecules comprising acassette containing the P. pastoris URA5 gene, or a homolog, variant orderivative thereof, flanked by direct repeat sequences. The directrepeat sequences are of sufficient length to mediate efficienthomologous recombination, thereby providing a means for deleting theURA5 marker from the host cell in preparation for another round oftransformation using the URA5 gene as a positive selection marker. Toincrease the efficiency of homologous recombination, the direct repeatsequences are preferably at least 200 nucleotides in length (see, e.g.,Wilson et al., Yeast; 16: 65-70 (2000)). Typically the direct repeatsequences are from around 200 nucleotides to around 1,100 nucleotides,but they may be even longer. In certain preferred embodiments, thedirect repeat sequences are derived from hisG segments of Salmonella.Alternatively, the direct repeats are obtained from segments of the lacZreading frame. One of skill in the art will readily appreciate thatvirtually any other direct repeat sequences may also be used to provideflanking sequences for recombination according to this aspect of theinvention.

The URA5-containing cassettes of the invention comprise URA5 sequenceswith flanking direct repeat sequences which mediate subsequent excissionof URA5 sequences from the host. Such URA5 cassettes allow for bothselection and counterselection for the URA5 gene activity. The positiveselection step is based on relieving auxotrophy to uracil, and thecounterselection is based on the acquisition of resistance to 5-FOA inuracil prototrophs. Boeke et al., Mol. Gen. Genet. 197:345-346 (1984).

Accordingly, the present invention provides a recombinant nucleic acidmolecule comprising a P. pastoris URA5 gene flanked by direct repeats(e.g., lacZ-URA5-lacZ, a “URA5 cassette”), which, upon expression,allows for selection and counterselection in a URA5⁻ host. In apreferred embodiment, yeast transformed with the P. pastoris URA5cassette have integrated the URA5 gene, e.g., into the host genome, at aselected location by homologous recombination between host andrecombinant nucleic acid sequences. Preferably, the host is deleted forendogenous URA5 sequences to discourage homologous recombination into anendogenous URA5 locus. The URA5 cassette-containing recombinant nucleicacid molecule preferably comprises sequences which target integration ofURA5 and other desired sequences into a select location of the yeasthost. As described, such transformants are selected on the basis ofconversion from Ura⁻ to Ura⁺ phenotypes. The direct repeats flanking theURA5 marker gene then facilitate homologous recombination events whichdelete the internal URA5 marker. Cells that have undergone such an eventrevert back to Ura⁻ and are selected by their ability to grow in thepresence of 5-FOA. This method provides for efficient, stableintegration of heterologous sequences into a host cell.

There are several advantages to using the P. pastoris URA5 markerselection of the present invention. First, this marker gene isrelatively small, only about 1 kb. The small size of the marker allowsfor construction of smaller plasmids. Moreover, the small size shouldreduce the rate of gene conversion of the auxotrophic marker gene duringtransformation in a Ura⁻ host strain which is not deleted for URA5sequences. This undesirable outcome can account for 10-50% oftransformed colonies in the case of the HIS4 marker. Higgins and Cregg,Meth. Mol. Biol., 103:1-15 (1998). A lower rate of gene conversionshould increase the fraction of transformants having knock-ins at thedesired target site. The P. pastoris URA5 marker gene of the inventionmay be used, moreover, to delete or otherwise disrupt endogenous URA5host sequences to further reduce the frequency of spontaneous reversionand hence false positive background colonies.

The isolated nucleic acid molecules of the instant invention mayadditionally include a sequence or gene of interest. As described above,a sequence or gene of interest typically encodes a protein that is notnormally produced in the host cell. In a preferred embodiment, yeasttransformed with the sequence or gene of interest have stably integratedthe sequence or gene of interest, e.g., into the host genome, at aselected location by homologous recombination between host andrecombinant nucleic acid sequences. The sequence or gene of interest maybe preferably linked to one or more expression control sequences, sothat the protein encoded by the sequence can be expressed underappropriate conditions in host cells that contain the isolated nucleicacid molecule.

The invention additionally provides isolated nucleic acid moleculesencoding a fragment of the P. pastoris SEC65 protein. The S. cerevisiaehomolog of this protein is related to mammalian SRP19, a subunit of thesignal recognition particle, and is thought to have similar function.Hann et al., Nature 356:532-533 (1992); Stirling and Hewitt, Nature356:534-537 (1992). Mutations in the S. cerevisiae SEC65 gene can causetemperature-sensitive cell growth and defects in the translocation ofseveral secreted and membrane-bound proteins. The S. cerevisiae SEC65protein is required for the stable association of another subunit,SRP54p, with the signal recognition particle. Id. Overexpression ofSRP54p suppresses both growth and protein translocation defects in cellscarrying a temperature-sensitive defect in the SEC65 gene. Nucleic acidmolecules encoding a fragment of the P. pastoris SEC65 gene can be usedto identify the full-length gene and can further be used to probe theexpression and functional activity of the encoded protein. Suchactivities may include structural and functional roles in the P.pastoris signal recognition particle and related effects on proteintranslocation across the endoplasmic reticulum. A shared extended domainstructure among fungal SEC65-encoded proteins and the ability oftruncation mutants of S. cerevisiae SEC65 to complement conditionallethal mutants in this gene (Regnacq et al., Mol Microbiol 29:753-762(1998)) indicate that polypeptides encoded by a fragment of the P.pastoris SEC65 gene may provide similar utility.

The invention further provides isolated nucleic acid molecules encodinga fragment of the P. pastoris SCS7 protein. Mutants of S. cerevisiaethat lack the S. cerevisiae homolog of SCS7 fail to accumulate aninositolphosphorylceramide species, IPC-C, which is the predominant formfound in wild-type cells. Dunn et al., Yeast 14:311-321 (1998). Instead,these mutants accumulate an IPC-B species believed to be unhydroxylatedon the amide-linked C26-fatty acid. In addition, elimination of the SCS7gene suppresses the Ca²⁺-sensitive phenotype of mutations in CSG1 andCSG2, genes required for mannosylation of IPC-C. Id. Accumulation ofIPC-C in cells carrying these mutations renders the cellsCa²⁺-sensitive. The full-length S. cerevisiae SCS7 gene encodes aprotein that contains both a cytochrome b5-like domain and a domain thatresembles the family of cytochrome b5-dependent enzymes that use ironand oxygen to catalyse desaturation or hydroxylation of fatty acids andsterols. Id. The encoded protein is therefore likely to be the enzymethat hydroxylates the C26-fatty acid of IPC-C. Effects of mutations inthe SCS7 gene on the lipid composition of a cell can be measured asdescribed in Haak et al., J. Biol. Chem. 272:29704-29710 (1997), whichis incorporated by reference herein in its entirety.

The isolated nucleic acid molecules encoding a fragment of the P. P.pastoris SCS7 protein of the present invention can be used to identifyand characterize the full-length form of the SCS7 gene. The isolatednucleic acid molecules of the invention can also be used to measureexpression of the SCS7 gene and to further characterize the structureand function of this gene and its encoded protein and the effects ofalterations in this gene on cellular metabolism.

Degenerate Oligonucleotides Useful for Cloning of P. pastoris URA5

In another embodiment, degenerate oligonucleotides useful in theisolation of the P. pastoris URA5 gene are provided. Theseoligonucleotides are capable of amplifying different portions of the P.pastoris URA5 gene. They can also bind to and amplify portions of the S.cerevisiae URA10 gene. That the oligonucleotides only amplify the URA5gene in P. pastoris suggests that this organism does not posses the URA10 gene. The oligonucleotides anneal to positions of the URA5 gene asshown in FIG. 3. Such oligonucleotides are also useful in hybridizationand amplification experiments.

Vectors

Also provided are vectors, including expression vectors, which comprisethe above nucleic acid molecules of the invention, as described furtherherein. In a first embodiment, the vectors include the isolated nucleicacid molecules described above. In an alternative embodiment, thevectors of the invention include the above-described nucleic acidmolecules operably linked to one or more expression control sequences.The vectors of the instant invention may thus be used to express apolypeptide having orotate-phosphoribosyl transferase activity.

The vectors of the invention may also include an element which ensuresthat they are stably maintained at a single copy in each cell (e.g., acentromere-like sequence such as “CEN”). Alternatively, the autonomouslyreplicating vector may optionally comprise an element which enables thevector to be replicated to higher than one copy per host cell (e.g., anautonomously replicating sequence or “ARS”). Methods in Enzymology, Vol.350: Guide to yeast genetics and molecular and cell biology, Part B.,Guthrie and Fink (eds.), Academic Press (2002).

In a preferred embodiment of the invention, the vectors arenon-autonomously replicating, integrative vectors designed to functionas gene disruption or replacement cassettes. An example of anintegrative vector of this type comprises at least at portion of aheterologous target gene linked to P. pastoris orotate-phosphoribosyltransferase (“OPT”)-encoding sequences which are preferably flanked bydirect repeat sequences. The vectors thus allow the targeted integrationof the sequences to be selected for by the expression of OPT activity incells carrying the integrated vectors. Subsequent excision of theOPT-encoding sequences is facilitated by the flanking direct repeatsequences.

In other embodiments, the integrative vectors of the invention mayinclude additionally heterologous sequences encoding proteins havingdesirable properties, e.g., those encoding glycosylation enzymes, sothat the desired sequences can be introduced into the host cell genomeas a result of the integration. These sequences remain in the host cellgenome even after the OPT-encoding sequences have been deleted byrecombination between flanking direct repeat sequences.

Isolated Polypeptides

According to another aspect of the invention, isolated polypeptides(including muteins, allelic variants, fragments, derivatives, andanalogs) encoded by the nucleic acid molecules of the invention areprovided. In one embodiment, the isolated polypeptide comprises thepolypeptide sequence corresponding to SEQ ID NOs:3, 5 or 7. In analternative embodiment of the invention, the isolated polypeptidecomprises a polypeptide sequence at least 65% identical to SEQ ID NOs:3,5 or 7. Preferably the isolated polypeptide of the invention has atleast 70%, 75% or 80% identity to SEQ ID NOs:3, 5 or 7. More preferably,the identity is 85%, 90% or 95%, but the identity to SEQ ID NOs:3, 5 or7 can be 98%, 99%, 99.9% or even higher.

According to other embodiments of the invention, isolated polypeptidescomprising a fragment of the above-described polypeptide sequences areprovided. These fragments preferably include at least 20 contiguousamino acids, more preferably at least 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100 or even more contiguous amino acids.

The polypeptides of the present invention also include fusions betweenthe above-described polypeptide sequences and heterologous polypeptides.The heterologous sequences can, for example, include heterologoussequences designed to facilitate purification and/or visualization ofrecombinantly-expressed proteins. Other non-limiting examples of proteinfusions include those that permit display of the encoded protein on thesurface of a phage or a cell, fusions to intrinsically fluorescentproteins, such as green fluorescent protein (GFP), and fusions to theIgG Fc region.

Host Cells

In another aspect of the invention, host cells transformed with thenucleic acid molecules or vectors of the invention, and descendantsthereof, are provided. In some embodiments of the invention, these cellscarry the nucleic acid sequences of the invention on vectors, which maybut need not be freely replicating vectors (see below). In otherembodiments of the invention, the nucleic acids have been integratedinto the genome of the host cells. In a preferred embodiment, the hostcells of the invention have been mutated by recombination with adisruption, deletion or mutation of the isolated nucleic acid of theinvention so that the activity of orotate-phosphoribosyl transferaseactivity in the host cell is reduced compared to a host cell lacking themutation. The host cell of the invention is preferably Pichia pastorisor Pichia methanolica, but other host cells, especially yeast cells, arealso encompassed within the scope of the invention.

In other embodiments of the invention, host cells defective inorotate-phosphoribosyl transferase activity are used to integrate one ormore sequences or genes of interest into the host cell genome usingnucleic acid molecules and/or methods of the invention. In someembodiments, the sequences or genes of interest are integrated so as todisrupt an endogenous gene of the host cell. Cells containing theintegration are identified by the recovery of uracil prototrophy due tothe concomitant integration of a gene encoding P. pastorisorotate-phosphoribosyl transferase. In a further embodiment of theinvention, uracil auxotrophs of the modified host cells are provided byselection of cells in which the P. pastoris orotate-phosphoribosyltransferase gene has been excised by homologous recombination.

Antibodies

In another aspect, the invention provides isolated antibodies, includingfragments and derivatives thereof, that bind specifically to theisolated polypeptides and polypeptide fragments of the present inventionor to one or more of the polypeptides encoded by the isolated nucleicacids of the present invention. The antibodies of the present inventionmay be specific for linear epitopes, discontinuous epitopes orconformational epitopes of such polypeptides or polypeptide fragments,either as present on the polypeptide in its native conformation or, insome cases, as present on the polypeptides as denatured, as, e.g., bysolubilization in SDS. Among the useful antibody fragments provided bythe instant invention are Fab, Fab′, Fv, F(ab′)₂, and single chain Fvfragments.

By “bind specifically” and “specific binding” is here intended theability of the antibody to bind to a first molecular species inpreference to binding to other molecular species with which the antibodyand first molecular species are admixed. An antibody is saidspecifically to “recognize” a first molecular species when it can bindspecifically to that first molecular species.

As is well known in the art, the degree to which an antibody candiscriminate as among molecular species in a mixture will depend, inpart, upon the conformational relatedness of the species in the mixture;typically, the antibodies of the present invention will discriminateover adventitious binding to unrelated polypeptides by at leasttwo-fold, more typically by at least 5-fold, typically by more than10-fold, 25-fold, 50-fold, 75-fold, and often by more than 100-fold, andon occasion by more than 500-fold or 1000-fold.

Typically, the affinity or avidity of an antibody (or antibody multimer,as in the case of an IgM pentamer) of the present invention for apolypeptide or polypeptide fragment of the present invention will be atleast about 1×10⁻⁶ M, typically at least about 5×10⁻⁷ M, usefully atleast about 1×10⁻⁷ M, with affinities and avidities of 1×10⁻⁸ M, 5×10⁻⁹M, 1×10⁻¹⁰ M and even stronger proving especially useful.

The isolated antibodies of the present invention may benaturally-occurring forms, such as IgG, IgM, IgD, IgE, and IgA, from anymammalian species. For example, antibodies are usefully obtained fromspecies including rodents—typically mouse, but also rat, guinea pig, andhamster—lagomorphs, typically rabbits, and also larger mammals, such assheep, goats, cows, and horses. The animal is typically affirmativelyimmunized, according to standard immunization protocols, with thepolypeptide or polypeptide fragment of the present invention.

Virtually all fragments of 8 or more contiguous amino acids of thepolypeptides of the present invention may be used effectively asimmunogens when conjugated to a carrier, typically a protein such asbovine thyroglobulin, keyhole limpet hemocyanin, or bovine serumalbumin, conveniently using a bifunctional linker. Immunogenicity mayalso be conferred by fusion of the polypeptide and polypeptide fragmentsof the present invention to other moieties. For example, peptides of thepresent invention can be produced by solid phase synthesis on a branchedpolylysine core matrix; these multiple antigenic peptides (MAPs) providehigh purity, increased avidity, accurate chemical definition andimproved safety in vaccine development. See, e.g., Tam et al., Proc.Natl. Acad. Sci. USA 85:5409-5413 (1988); Posnett et al., J. Biol. Chem.263, 1719-1725 (1988).

Protocols for immunization are well-established in the art. Suchprotocols often include multiple immunizations, either with or withoutadjuvants such as Freund's complete adjuvant and Freund's incompleteadjuvant. Antibodies of the present invention may be polyclonal ormonoclonal, with polyclonal antibodies having certain advantages inimmunohistochemical detection of the proteins of the present inventionand monoclonal antibodies having advantages in identifying anddistinguishing particular epitopes of the proteins of the presentinvention.

Following immunization, the antibodies of the present invention may beproduced using any art-accepted technique. Host cells for recombinantantibody production—either whole antibodies, antibody fragments, orantibody derivatives—can be prokaryotic or eukaryotic. Prokaryotic hostsare particularly useful for producing phage displayed antibodies, as iswell known in the art. Eukaryotic cells, including mammalian, insect,plant and fungal cells, are also useful for expression of theantibodies, antibody fragments, and antibody derivatives of the presentinvention. Antibodies of the present invention can also be prepared bycell free translation.

The isolated antibodies of the present invention, including fragmentsand derivatives thereof, can usefully be labeled. It is, therefore,another aspect of the present invention to provide labeled antibodiesthat bind specifically to one or more of the polypeptides andpolypeptide fragments of the present invention. The choice of labeldepends, in part, upon the desired use. In some cases, the antibodies ofthe present invention may usefully be labeled with an enzyme.Alternatively, the antibodies may be labeled with colloidal gold or witha fluorophore. For secondary detection using labeled avidin,streptavidin, captavidin or neutravidin, the antibodies of the presentinvention may usefully be labeled with biotin. When the antibodies ofthe present invention are used, e.g., for Western blotting applications,they may usefully be labeled with radioisotopes, such as ³³P, ³²P, ³⁵S,³H and ¹²⁵I. As would be understood, use of the labels described aboveis not restricted to any particular application.

Methods for the Genetic Integration of Nucleic Acid Sequences:Disruption of a Host Gene Encoding Orotate-Phosphoribosyl Transferase

According to another embodiment of the instant invention, a method forthe genetic integration of a heterologous nucleic acid sequence into thegenome of a host cell is provided. In one aspect of this embodiment, ahost gene encoding orotate-phosphoribosyl transferase is disrupted bythe introduction of a disrupted, deleted or otherwise mutated nucleicacid sequence derived from the P. pastoris URA5 gene disclosed herein.Accordingly, disrupted host cells having a point mutation,rearrangement, insertion or preferably a deletion (including a “markeddeletion”, in which a heterologous selectable sequence has replaced thedeleted URA5 sequence) are provided. Host cells disrupted in the URA5gene and consequently lacking in orotate-phosphoribosyl transferaseactivity serve as suitable hosts for further embodiments of theinvention in which heterologous sequences may be introduced into thehost cell genome by targeted integration.

Methods for the Genetic Integration of Nucleic Acid Sequences:Introduction of a Sequence of Interest in Linkage with a Marker Sequence

In another aspect of the instant invention, a heterologous nucleic acidsequence is introduced into a yeast host cell lackingorotate-phosphoribosyl transferase (OPT) activity (i.e., Ura5⁻). Theheterologous nucleic acid sequences introduced using this method arelinked to a nucleic acid sequence that encodes the P. pastoris OPTactivity, preferably on a vector. Upon transformation of the vector intocompetent Ura5⁻ host cells, cells containing heterologous sequenceslinked to the OPT-encoding sequences of the invention may be selectedbased on their ability to grow in the absence of added uracil.

In one embodiment, the method comprises the step of introducing into acompetent Ura5⁻ host cell an autonomously replicating vector which ispassed from mother to daughter cells during cell replication. Theautonomously replicating vector comprises heterologous nucleic acidsequences of interest linked to P. pastoris OPT-encoding sequences andoptionally comprises an element which ensures that it is stablymaintained at a single copy in each cell (e.g., a centromere-likesequence such as “CEN”). In another embodiment, the autonomouslyreplicating vector may optionally comprise an element which enables thevector to be replicated to higher than one copy per host cell (e.g., anautonomously replicating sequence or “ARS”).

In a preferred embodiment, the vector is a non-autonomously replicating,integrative vector which is designed to function as a gene disruption orreplacement cassette. An integrative vector of the invention comprisesone or more regions comprising “target gene sequences” (sequences whichcan undergo homologous recombination with sequences at a desired genomicsite in the host cell) linked to P. pastoris OPT-encoding sequences ofthe invention which are preferably flanked by direct repeat sequences(see below). The OPT-encoding sequences may be adjacent to the targetgene sequences (e.g., a gene replacement cassette) or may be engineeredto disrupt the target gene sequences (e.g., a gene disruption cassette).The presence of target gene sequences in the replacement or disruptioncassettes targets integration of the cassette to specific genomicregions in the host by homologous recombination.

In a preferred method of the invention, a host gene that encodes anundesirable activity, (e.g., an enzymatic activity) may be mutated(e.g., interrupted) by targeting a P. pastoris OPT-encoding replacementor disruption cassette of the invention into the host gene by homologousrecombination. In a preferred embodiment, an undesired glycosylationenzyme activity (e.g., an initiating mannosyltransferase activity suchas OCH1) is disrupted in the host cell to alter the glycosylation ofpolypeptides produced in the cell.

Preferably, the target gene replacement or disruption cassette of theinvention further comprises direct repeat sequences flanking the P.pastoris orotate-phosphoribosyl transferase gene. The properties of suchdirect repeat sequences have already been described. After targetedintegration of the cassette into the host cell genome and selection ofintegrants for growth in the absence of uracil, the direct repeatsequences flanking the orotate-phosphoribosyl transferase gene promotethe excision of the OPT-encoding gene out of the host genome. Cellslacking orotate-phosphoribosyl transferase activity are convenientlycounterselected for their ability to grow in medium containing 5-FOA.One of skill in the art would appreciate, however, that other means maybe used to counterselect for cells lacking orotate-phosphoribosyltransferase activity. Because the cells obtained from thecounterselection step lack orotate-phosphoribosyl transferase activity,the same P. pastoris OPT-encoding nucleic acid sequence may be used inrepeated gene disruption events according to this aspect of theinvention.

In yet a further embodiment of the invention, a gene encoding aheterologous protein is engineered in linkage to the P. pastoris URA5gene within the gene replacement or disruption cassette. Ina preferredembodiment, the cassette is integrated into a locus of the host genomewhich encodes an undesirable activity, such as an enzymatic activity.For example, in one preferred embodiment, the cassette is integratedinto a host gene which encodes an initiating mannosyltransferaseactivity such as the OCH1 gene. In a more preferred embodiment, thecassette further comprises one or more genes encoding desirableglycosylation enzymes, including but not limited tomannosyltransferases, N-acetylglucosaminyltransferases (GnTs),UDP-N-acetylglucosamine transporters, galactosyltransferases (GalTs),sialyltransferases (STs) and protein-mannosyltransferases (PMTs). Inanother preferred embodiment, the cassette comprises one or more genesencoding useful therapeutic proteins, e.g., kringle domains of the humanplasminogen, erythropoietin, cytokines such as, but not limited to,interferon-α, interferon-β, interferon-γ, interferon-ω, andgranulocyte-CSF, coagulation factors such as factor VIII, factor IX, andhuman protein C, soluble IgE receptor α-chain, IgG, IgG fragments, IgM,urokinase, chymase, and urea trypsin inhibitor, IGF-binding protein,epidermal growth factor, growth hormone-releasing factor, annexin Vfusion protein, angiostatin, vascular endothelial growth factor-2,myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1antitrypsin, DNase II and α-feto proteins. The engineered cassette isuseful for “knocking-in” genes encoding such glycosylation enzymes andother sequences of interest in strains of yeast cells to produceglycoproteins with human-like glycosylations and other useful proteinsof interest. Representative methods for producing human-likeglycoproteins are described in WO 02/00879 and are incorporated byreference herein.

The following examples are for illustrative purposes and are notintended to limit the scope of the invention.

Example 1 General Materials and Methods

Escherichia coli strain DH5α (Invitrogen, Carlsbad, Calif.) was used forrecombinant DNA work. P. pastoris strains NRRL Y-11430 (wild-type) andJC308 (ade1 arg4 his4 ura3) (Lin Cereghino et al., Gene 263:159-169(2001)) were used for construction of yeast strains. PCR reactions wereperformed according to supplier recommendations using either ExTaq(TaKaRa, Madison, Wis.), Taq Poly (Promega, Madison, Wis.) or Pfu Turbo(Stratagene, Cedar Creek, Tex.). Restriction and modification enzymeswere from New England Biolabs (Beverly, Mass.) or Promega.

PCR analysis of the modified yeast strains was as follows. A singlecolony was resuspended in 100 μl breaking buffer (100 mM NaCl, 10 mMTris, pH 8.0, 1 mM EDTA). After addition of 100 mg of acid washed glassbeads and 100 μl of phenol-chloroform, the solution was vortexed for 1min. The mixture was then centrifuged for 5 min at full speed in amicrocentrifuge, the supernatant recovered, and the genomic DNA wasprecipitated by addition of 1 ml ice cold ethanol. Following a wash with70% ethanol, the pellet was resuspended in 10 μl breaking buffer, and0.5 to 1 μl were used for PCR analysis.

Cloning of the P. pastoris URA5 Gene

Several strategies may be used for cloning and identifying the P.pastoris URA5 gene. A preferred method involves using the sequencehomology of the existing S. cerevisiae URA5 gene in combination withconservation of gene order in a variety of yeast species. Two genes,URA5 and SEC65, are located adjacent to one another in oppositeorientations in at least four yeast species: S. cerevisiae, K. lactis,C. albicans and Y. lipolytica. Sánchez and Domínguez, Yeast 18:807-813(2001). Protein sequences encoded by each of these genes are known inthese and other microorganisms (FIG. 2), and these sequences were usedto design degenerate primers, e.g., using the CODEHOP strategy. Rose etal., Nucleic Acids Res. 26:1628-1635 (1998). Two such primers,designated URA5-1 (FIG. 3) (SEQ ID NO:23) and Sec65-1(AAGAGATTTCAAGTTTTGTACCCADKNTAYTTYGA) (SEQ ID NO:29), were used toamplify a 1.1 kb DNA fragment from P. pastoris genomic DNA. URA5-1 is onthe top strand starting from amino acid 27. This PCR fragment was thencloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) andsequenced.

The 1100 bp fragment generated by PCR shows high homology on one end toURA5 of S. cerevisiae and on the other end to SEC65 genes from S.cerevisiae, K. lactis, Y. lipolytica, and S. pombe. The derivednucleotide sequence was used to search the partial genomic sequence ofP. pastoris, as provided by Integrated Genomics, Inc. (Chicago, Ill.).Results of this search identified an overlapping DNA fragment thatincludes an additional 0.9 kb DNA sequence adjacent to the primer site.Within this sequence is the predicted initiation codon for proteintranslation. The predicted initiation codon is preceded by about 150nucleotides of upstream regulatory sequences (including promotorsequences) and about 0.7 kb of the 3′ region of a gene with highhomology to S. cerevisiae SCS7 (FIG. 1). The protein sequence derived bytranslation of the P. pastoris URA5 gene shows about 64% identity andabout 78% similarity to the URA5 gene from S. cerevisiae, and alsodisplays high homology to URA5 genes from other species. The complete1947 bp fragment is shown in FIG. 1.

Cloning of P. pastoris URA5 Using Alternative DegenerateOligonucleotides

Degenerate primers were designed using the CODEHOP strategy. Rose etal., Nucleic Acids Res. 26:1628-1635 (1998). URA5-1 (SEQ ID NO:23) is adegenerate form of the coding strand, starting from the codon encodingamino acid 27. URA5-2 (SEQ ID NO:24) is a degenerate form of the codingstrand starting from the codon encoding amino acid 66. URA5-3 (SEQ IDNO:25) is the partial complement of URA5-2. URA5-4 (SEQ ID NO:26) is adegenerate form of the coding strand, starting from the codon encodingamino acid 105. URA5-5 (SEQ ID NO:27) is the partial complement ofURA5-4. URA5-6 (SEQ ID NO:28) is a degenerate form of the non-codingstrand, designed to hybridize to the segment of the coding strandstarting at the codon encoding amino acid 130. The sequence of andpositions within URA5 bound by the oligonucleotides are illustrated inFIG. 3.

Example 2 Disruption of the P. pastoris URA5 Gene

The cloned URA5 gene, together with gene-specific primers, may be usedto generate a construct to disrupt the URA5 gene from the genome of P.pastoris. Host cells with a disrupted URA5 gene were created using a P.pastoris URA5 disruption cassette as follows.

A 1.5 kb SacI, XbaI fragment containing the kanamycin-resistance gene oftransposon Tn903 was excised from plasmid pUG6 (Güldener et al., NucleicAcids Res. 24:2519-2524 (1996)) and cloned into the SacI, XbaI sites ofpUC19 (New England Biolabs, Beverly, Mass.) resulting in pJN374.Oligonucleotides Ura5-55 (GGGATATCGGCCTTTGTTGATGCAAGTTTTACGTGGATC) (SEQID NO:30) and Ura5-53p (GCGATATCGGTGAAAGTTCCAAACTTCAAGGCCTGCGAAG) (SEQID NO:31) were used to amplify a region upstream of the URA5 ATG usingP. pastoris genomic DNA as a template and Pfu Turbo DNA polymerase. Theresulting DNA fragment was cut with EcoRV and cloned into the EcoICRIsite of pJN374. The resulting plasmid was then digested with SalI andSphI. A DNA fragment corresponding to part of the coding sequence andthe 3′ region of the URA5 gene was amplified using oligonucleotidesUra5-35P (GACGCGTCGACGGTCTTTTCAACAAAGCTCCATTAGTGAG) (SEQ ID NO:32) andUra5-33 (ACATGCATGCGCCAAAAGGAGTATGGTGTGGAGAACCC) (SEQ ID NO:33). Thisfragment was inserted into the cut plasmid to create pJN395 (FIG. 5). Inthis plasmid, codons 27 to 39 of the P. pastoris URA5 gene have beenreplaced by the kanamycin-resistance gene.

Following digestion of pJN395 with EcoRI and SphI, the linearizeddisruption cassette was transformed into P. pastoris wild type strainNRRL Y-11430 (ATCC 76273) by electroporation, and the cells were platedonto YPD plates (Methods in Enzymology, Vol. 350: Guide to yeastgenetics and molecular and cell biology, Part B., Guthrie and Fink(eds.), Academic Press (2002)) containing 300 mg/l Geneticin(Invitrogen, Carlsbad, Calif.). After 4 days of incubation at 30° C.,approximately 10,000 clones were replicated onto plates containing 5-FOA(1.4% Yeast Nitrogen Base, 2% Dextrose, 0.2 g/l Uracil, 1 g/15-FOA, 4mg/l Biotin, 1.5% Agar) and incubated for 7 days at 30° C. Coloniesresistant to 5-FOA (240 colonies) were restreaked once on 5-FOA platesand then patched onto YPD and Ura dropout plates (Methods in Enzymology,Vol. 350: Guide to yeast genetics and molecular and cell biology, PartB., Guthrie and Fink (eds.), Academic Press (2002)). Cells that failedto grow on Ura dropout plates but that were able to grow on YPD (205 ofthe original 240 resistant colonies) were then amplified in liquid YPD.Approximately 10⁸ cells of each were plated onto single Ura dropoutplates to check for revertants. Thirteen clones gave rise to colonies onthe Ura dropout plates and were not examined further. The other cloneswere unable to revert spontaneously. Based on the most robust growth onYPD and 5-FOA, thirty-one clones were picked and examined by colony PCR,of which, thirty were found to be kanamycin-marked URA5 knockouts. Toconfirm that the genomic URA5 gene in these strains was disrupted withthe kanamycin-resistance gene, one of the colony PCR reactions wasdigested with either BglII or XbaI. A strain displaying the expectedrestriction pattern (fragments of 2.35 kb and 1.05 kb) was designatedYJN165.

Example 3 Construction of a Set of Vectors for the Stable GeneticModification of P. pastoris

A set of vectors useful for stable gene replacement in Pichia pastoriswas constructed as described below. Based on the high copy vector pUC19(Yanisch-Perron et al., Gene 33:103-119 (1985)), a set of modularplasmids was assembled that, after a few simple subcloning steps, may beused to replace any P. pastoris gene with a heterologous gene ofinterest under the control of the strong P. pastoris GAPDH promotor.Plasmid pJN266 (FIG. 4) consists of two fragments homologous to the 5′and 3′ regions of the P. pastoris KEX1 gene. These segments flank a P.pastoris GAPDH promotor, a S. cerevisiae CYC1 transcriptional terminatorexpression cassette (“CYC1 TT”) and a S. cerevisiae URA3 auxotrophicmarker cassette. All regions of this plasmid are flanked by multiplerestriction sites and can be individually replaced. The expressioncassette contains a multiple cloning site for the insertion ofheterologous genes.

Two reusable auxotrophic marker cassettes were constructed based on theapproach described by Lu et al., Appl. Microbiol. Biotechnol. 49:141-146(1998) and Alani et al., Genetics 116:541-545 (1987), using directrepeats from segments of the lacZ reading frame as recombination sites.As counterselectable auxotrophic markers, a 2 kb DNA fragment containingthe P. pastoris URA3 gene or a 1 kb fragment harboring the P. pastorisURA5 gene were used. Both marker cassettes were then inserted into a P.pastoris OCH1 knockout plasmid. The P. pastoris URA5-containing plasmidwas then modified further to generate a plasmid that includes theheterologous gene for the UDP-N-acetylglucosamine transporter of K.lactis.

Methods

The first step in plasmid construction involved creating a set ofuniversal plasmids containing DNA regions of the KEX1 gene of P.pastoris (Boehm et al., Yeast 15:563-572 (1999)) as space holders forthe 5′ and 3′ regions of the genes to be knocked out. The plasmids alsocontained the S. cerevisiae URA3 gene, flanked by bacterial directrepeat sequences (Alani et al., Genetics 116: 541-545 (1987)) as a spaceholder for the auxotrophic markers and an expression cassette with amultiple cloning site for insertion of a foreign gene.

A 0.9-kb fragment of the P. pastoris KEX1-5′ region was amplified by PCRusing primers Kex 55 (GGCGAGCTCGGCCTACCCGGCCAAGGCTGAGATCATTTGTCCAGCTTCAGA) (SEQ ID NO:34) and Kex 53(GCCCACGTCGACGGATCCGTTTAAACATCGATTGGAGAGGCTGACACC GCTACTA) (SEQ IDNO:35) with P. pastoris genomic DNA as a template. The amplifiedfragment was cloned into the SacI, SalI sites of pUC19 (New EnglandBiolabs, Beverly, Mass.). The resulting plasmid was cut with BamHI andSalI. A 0.8-kb fragment of the KEX1-3′ region that had been amplifiedusing primers Kex 35 (CGGGATCCACTAGTATTTAAATCATATGTGCGAGTGTACAACTCTTCCCACATGG) (SEQ ID NO:36) and Kex 33(GGACGCGTCGACGGCCTACCCGGCCGTACGAGGAATTTCTCGGATGAC TCTTTTC) (SEQ IDNO:37) with P. pastoris genomic DNA as a template was cloned into thecut plasmid to create pJN262. This plasmid was further cut with BamHI.The 3.8-kb BamHI, BglII fragment from pNKY51 (Alani et al., Genetics116:541-545 (1987)) was then inserted into this site in both possibleorientations to generate pJN263 and pJN264.

An expression cassette was created using NotI and PacI as cloning sites.The GAPDH promoter of P. pastoris was amplified using primers Gap 5(CGGGATCCCTCGAGAGATCTTTTTTGTAGAAATGTCTTGGTGCCT) (SEQ ID NO:38) and Gap 3(GGACATGCATGCACTAGTGCGGCCGCCACGTGATAGTTGTTCA ATTGATTGAAATAGGGACAA) (SEQID NO:39) with the plasmid pGAPZ-A (Invitrogen, Carlsbad, Calif.) as atemplate. The amplified segment was cloned into the BamHI, SphI sites ofpUC19 (New England Biolabs, Beverly, Mass.). The resulting plasmid wascut with SpeI and SphI. The CYC1 transcriptional terminator region wasamplified using primers Cyc 5(CCTTGCTAGCTTAATTAACCGCGGCACGTCCGACGGCGGCCCACGGGT CCCA) (SEQ ID NO:40)and Cyc 3 (GGACATGCATGCGGATCCCTTAAGAGCCGGCAGCTTGCAAATTAAAGCCTTCGAGCGTCCC) (SEQ ID NO:41) with plasmid pPICZ-A (Invitrogen,Carlsbad, Calif.) as a template. The amplified segment was cloned intothe cut plasmid to create pJN261. The expression cassette was generatedby digestion of this plasmid with BamHI. This fragment was cloned eitherinto pJN263 (supra) to generate plasmid, pJN265, or into pJN264 (supra)to generate plasmids pJN266 and pJN267, depending on orientation of theinsert. The map of pJN266 is shown in (FIG. 4).

A knockout plasmid for the P. pastoris OCH1 gene was created bydigesting pJN263 with SalI and SpeI. A 2.9 kb DNA fragment of theOCH1-5′ region, amplified using the primers Och55(GAACCACGTCGACGGCCATTGCGGCCAAAACCTTTTTTCCTATTCAAA CACAAGGCATTGC) (SEQ IDNO:42) and Och 53 (CTCCAATACTAGTCGAAGATTATCTTCTACGGTGCCTGGACTC) (SEQ IDNO:43) with P. pastoris genomic DNA as a template, was cloned into theopen sites. The resulting plasmid was cut with EcoRI and PmeI A 1.0-kbDNA fragment of the OCH1-3′ region, amplified using the primers Och 35(TGGAAGGTTTAAACAAAGCTAGAGTAAAATAGATATAGCGAGATTAG AGAATG) (SEQ ID NO:44)and Och 33 (AAGAATTCGGCTGGAAGGCCTTGTACCTTGATGTAGTTCCCGTTTTCAT C) (SEQ IDNO:45) with P. pastoris genomic DNA as a template was inserted into thecut plasmid to generate pJN298. To allow for the possibility ofsimultaneously knocking out the OCH1 gene and introducing a new gene,the BamHI expression cassette of pJN261 (supra) was cloned into theunique BamHI site of pJN298 to create pJN299.

The P. pastoris gene disruption cassettes for URA3 and URA5 wereconstructed using a strategy similar to that described in Lu et al.,Appl. Microbiol. Biotechnol. 49:141-146 (1998). A 2.0-kb PstI, SpeIfragment of the P. pastoris URA3 gene was inserted into the PstI, XbaIsites of pUC19 (New England Biolabs, Beverly, Mass.) to create pJN306. A0.7-kb SacI, PvuII DNA fragment of the lacZ open reading frame from E.coli (see, e.g., Kalnins et al., EMBO J. 2:593-597 (1983)) was clonedinto the SacI, SmaI sites to yield pJN308. Following digestion of pJN308with PstI and treatment with T4 DNA polymerase, the SacI-PvuII fragmentfrom lacZ, blunt-ended with T4 DNA polymerase, was inserted into theplasmid to generate pJN315 (FIG. 4). The lacZ/URA3 disruption cassettewas released by digestion of pJN315 with SacI and SphI and blunt-endedwith T4 DNA polymerase. The cassette fragment was then cloned into thebackbone of pJN299 (supra) that had been digested with PmeI and AflIIand blunt-ended with T4 DNA polymerase. The resulting plasmid was namedpJN329 (FIG. 4). See also Choi et al., Proc. Natl. Acad. Sci. USA100:5022-5027 (2003).

To generate a lacZ/URA5 disruption cassette, the SacI, PvuII fragment oflacZ was cloned into the SacI, SmaI sites of PUC19. The resultingplasmid was digested with PstI and blunted and the lacZ fragment thathad been blunt-ended using T4 DNA polymerase was inserted into theplasmid to yield pJN316. A 1.0 kb fragment of the P. pastoris URA5 genewas amplified from the genomic DNA using primers Ura5Comp5(GCTCTAGAGGGACTTATCTGGGTCCAGACGATGTG) (SEQ ID NO:46) and Ura5Comp3(CGGGATCCGCCGCCGTGCCCAAAGCTCCGAAACAG) (SEQ ID NO:47) and cloned into theBamHI, XbaI sites of pJN316 to generate pJN396 (FIG. 5). The lacZ/URA5cassette was released by digestion of this plasmid with EcoRI and SphI.

To create OCH1 knockout plasmids containing different auxotrophicmarkers, pJN299 (supra) was digested with PmeI and AflII and treatedwith T4 DNA polymerase. Following digestion of pJN315 (FIG. 4) with SacIand SphI, and digestion of pJN396 (FIG. 5) with EcoRI and SphI, each ofthe auxotrophic marker cassettes was blunt-ended with T4 DNA polymeraseand ligated into the pJN299 backbone. This yielded plasmids pJN329(URA3) and pJN398a (URA5), respectively.

Plasmid pJN398 was further modified by digestion with SpeI and NotI andblunt ended using T4 DNA polymerase. A blunt-ended BglII/HindIIIfragment of pDL02 derived from Genbank Accession AF106080 (Abeijon etal., Proc. Natl. Acad. Sci. USA 93:5963-5968 (1996)) and containing theUDP-N-acetylglucosamine transporter of K. lactis was cloned into theopen sites to create pJN407 (FIG. 5).

Example 4 Disruption of the P. pastoris OCH1 Gene and Regeneration ofCounterselectable Markers

The disruption of P. pastoris OCH1 in strain JC308 (ade1, arg4, his4,ura3) (Lin Cereghino et al., Gene 263:159-169 (2001)) using plasmidpJN329 (URA3) has been described in Choi et al. Proc. Natl. Acad. Sci.USA, 100:5022-5027 (2003), which is hereby incorporated by reference inits entirety.

To replace the P. pastoris OCH1 gene with the gene for theUDP-N-acetylglucosamine transporter of K. lactis using the URA5counterselectable marker, 100 μg of pJN407 was digested with SfiI andtransformed by electroporation into YJN165. Following incubation onminimal medium lacking uracil for ten days at room temperature, 460colonies were picked and re-streaked. After three days, all 460 cloneswere streaked onto two sets of YPD plates. The five URA⁺ clones thatwere unable to grow at 37° C., but that grew at room temperature, weresubjected to colony PCR to test for the deletion of the P. pastoris OCH1gene. All five strains gave rise to a PCR signal of the expected size. Asecond colony PCR confirmed that all five clones also contained the genefor the K. lactis UDP-N-Acetyl-Glucosamine Transporter. These cloneswere designated YJN198-1 through 5.

To regenerate the ura5 auxotroph, all five clones containing theoch1::URA5 allele were grown on YPD plates for two days and then spreadonto 5-FOA plates. After six days of incubation at room temperature, allfive clones gave rise to colonies that were resistant to 5-FOA and thatwere also auxotrophic for uracil. The colonies resistant to 5-FOA thatwere derived by counterselection of YJN198-2 and YJN198-3 grewsignificantly slower than the others on YPD and were not examinedfurther. The six fastest growing colonies that were derived bycounterselection of YJN198-1, YJN198-4 and YJN198-5 were subjected toPCR analysis. These colonies were all confirmed to have lost the URA5cassette. They were designated YJN199-1 through 6.

A schematic of the disruption and marker recycling steps occurring inthe stable integration of the UDP-GlcNAc Transporter into the OCH1 locususing the P. pastoris URA5-K. lactis UDP-GlcNAc Transporter cassette isshown in FIG. 6.

While preferred illustrative embodiments of the present invention aredescribed, one skilled in the art will appreciate that the presentinvention can be practiced by other than the described embodiments,which are presented for purposes of illustration only and not by way oflimitation. The present invention is limited only by the claims thatfollow.

1-26. (canceled)
 27. An isolated antibody or antigen-binding fragment orderivative thereof which binds selectively to the isolated polypeptideof claim 12, 14 or
 15. 28. A method for the genetic integration of aheterologous nucleic acid sequence in a host cell comprising the step ofdisrupting a host gene encoding orotate-phosphoribosyl transferase byintroduction of a disrupted, deleted or mutated nucleic acid sequencederived from a sequence selected from the group consisting of: (a) SEQID NO:2; (b) a nucleic acid sequence that is a degenerate variant of SEQID NO:2; (c) a nucleic acid sequence at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99% or at least 99.9% identical to SEQ ID NO:2; (d)a nucleic acid sequence that encodes a polypeptide having the amino acidsequence of SEQ ID NO:3; (e) a nucleic acid sequence that encodes apolypeptide at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99% or atleast 99.9% identical to SEQ ID NO:3; (f) a nucleic acid sequence thathybridizes under stringent conditions to SEQ ID NO:2; and (g) a nucleicacid sequence comprising a fragment of any one of (a)-(f) that is atleast 60 contiguous nucleotides in length.
 29. A method for the geneticintegration of a heterologous nucleic acid sequence in a host celllacking orotate-phosphoribosyl transferase activity comprising the stepof introducing a sequence of interest into the host cell in linkage witha sequence encoding orotate-phosphoribosyl transferase activity selectedfrom the group consisting of: (a) SEQ ID NO:2; (b) a nucleic acidsequence that is a degenerate variant of SEQ ID NO:2; (c) a nucleic acidsequence at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99% or atleast 99.9% identical to SEQ ID NO:2; (d) a nucleic acid sequence thatencodes a polypeptide having the amino acid sequence of SEQ ID NO:3; (e)a nucleic acid sequence that encodes a polypeptide at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99% or at least 99.9% identical to SEQID NO:3; (f) a nucleic acid sequence that hybridizes under stringentconditions to SEQ ID NO:2; and (g) a nucleic acid sequence comprising afragment of any one of (a)-(f) that is at least 60 contiguousnucleotides in length.
 30. The method of claim 29, wherein theheterologous nucleic acid sequence is stably integrated.
 31. The methodof claim 29, wherein the sequence encoding orotate-phosphoribosyltransferase activity is flanked by direct repeat sequences.
 32. Themethod of claim 29, wherein the sequence of interest encodes apolypeptide.
 33. The method of claim 32, wherein the polypeptide is aglycosylation enzyme.
 34. The method of claim 33, wherein theglycosylation enzyme is selected from the group consisting ofglycosidases, mannosidases, phosphomannosidases, phosphatases,nucleotide sugar transporters, mannosyltransferases, theN-acetylglucosaminyltransferases, the UDP-N-acetylglucosaminetransporters, the galactosyltransferases, the sialyltransferases and theprotein mannosyltransferases.
 35. The method of claim 32, wherein thepolypeptide is a therapeutic protein.
 36. The method of claim 35,wherein the therapeutic protein is selected from the group consisting ofkringle domains of the human plasminogen, erythropoietin, cytokines,coagulation factors, soluble IgE receptor α-chain, IgG, IgG fragments,IgM, urokinase, chymase, urea trypsin inhibitor, IGF-binding protein,epidermal growth factor, growth hormone-releasing factor, annexin Vfusion protein, angiostatin, vascular endothelial growth factor-2,myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1antitrypsin, DNase II and α-feto proteins.
 37. The method of claim 31,further comprising the step of counterselecting for loss oforotate-phosphoribosyl transferase activity.
 38. The method of claim 37,wherein the counterselection is for growth on 5-fluoroorotic acid. 39.The method of claim 29, wherein the heterologous nucleic acid sequenceis introduced into an OCH1 locus of the host cell.